Development Of Aspergillus Oryzae Auxotroph Expression System And Application In Expressing A Cold-adapted ?-Amylase From Psychrotolerant Fungi

Aspergillus oryzae is a filamentous fungus, Generally Recognized As Safe (GRAS), producing various enzymes. It is used extensively in the manufacture of fermented foods for over 1,000 years. Screening of Aspergillus oryzae auxotroph and construction of relevant transformation systerms have been studied for about three decades. A. oryzae has also been considered a favorable host for enzyme production by virtue of its ability to secrete proteins towards the extracellular medium and eukaryotic post-translational modifications.Amylase is a biocatalyst that can degrade starch into oligosaccharides, widely used in food, detergent, medicine, and industrial fermentation. One of the most important amylase is a-amylase. It holds above 25% of enzyme market in present. The a-amylases applied in industry are mainly bacterial thermostable a-amylase and fungal medium temperature a-amylase. Nowadays, cold-adapted amylase is still under studying which is mostly derived from bacteria.In this study, ultraviolet mutagenesis of wild strain A. oryzae RIB40 was conducted, resulting in six uracil auxotroph mutants which were screened on the 5-FOA agar plates. Through molecular biology identification, five mutants were pyrF auxotroph (FP1, FP4, FP6, PF1, PF3) and the other was pyrG auxotroph (PF4). Complementation experiment was carried out with vector harbouring pyrF or pyrG. It turned out that FP1, FP2, FP3, PF1, PF3 could be only complemented with pyrF, while PF4 just met this condition with pyrG. It suggests that the pyrF and pyrG are two sEParate genes in A. oryzae, and the corresponding encoded proteins, OPRTase and OMPdecase respectively, are two sEParate functional enzymes, which is similar as in the bacteria and yeast but different with the animal and plant.Expression plasmid pBC12FNH was constructed by inserting promoter and terminator of A. oryzae amyB, signal pEPtide, pyrF and His-tag label. Then GFP was introduced, and the recombinant plasmid was transformed into A. oryzae. Then GFP protein could be detected after induced cultivation, indicating that A. oryzae pyrF expression system was successfully founded.Geomyces pannorum R1-2 a-amylase gene was cloned by two round degenerate PCRs, resulting a fragment with a length of 933 bp. The whole length of a-amylase gene was obtained by gene walking and reverse transcription PCR. The cDNA is composed of 1,497 nucleotides, which deduced protein includes 494 amino acid residues. The cloned gene was defined as GpA2. It shares the highest amino acid identity (58%) with the ?-amylase from Lipomyces kononenkoae. Then GpA2 was expressed in A. oryzae expression at 20?. Transformant, NHA2-1, was screened by enzyme activity detection and verified by western blot. The curves of growth and enzyme production of NHA2-1 and RIB40 were studied. It turned out that NHA2-1 grew better than the control at earlier stage, implying that the dextrin was hydrolyzed more effectively by heterologous cold-active amylase (GpA2) at low temperature. Otherwise, the enzyme production of NHA2-1 reached a balance at the 4th day while that of the wild could be approximately at the 5th day. The highest enzyme production of NHA2-1 was 958 U/mL,1.72-fold higher than that of the wild. The amylase activities of the transformant NHA2-1 retained about 86.6% of the maximal activities at 40?, while the parent strains retained only 67.0%.The recombinant ?-amylase was purified by affinity chromatography and a single protein band of approximate 54 kDa was observed. The specific activity of purified GpA2 was 9.72×103 U/mg, and kinetic parameters were 3.217 mg/mL (Km) and 3.328×10-2 mg/(mL·min) (Vmax) towards soluble starch, respectively. The optimum temperature and pH of GpA2 were 40? and 5.0, respectively. Mg2+ and Ca2+ increased the activity of the purified GpA2. Catalytic efficiency of the purified GpA2 was increased by Mg2+ and Ca2+, while it was obviously inhibited by Ni2+, surfactants (Tween-80, Tween-20 and Triton X-100) and methanol. The purified GpA2 exhibited broad substrate specificity that could efficiently hydrolyze all the substrates tested excEPt pullulan. The main hydrolysis products of soluble starch catalyzed by the purified GpA2 were glucose, maltose and maltotriose with no detection of maltotetraose and other oligosaccharides, showing its prospect of application in the starch syrup industry.